title: Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.
abstract: Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Pragya
authors.initials: P
authors.lastname: Singh
authors.forename: Scott A
authors.initials: SA
authors.lastname: Shaffer
authors.forename: Alexander
authors.initials: A
authors.lastname: Scherl
authors.forename: Carol
authors.initials: C
authors.lastname: Holman
authors.forename: Richard A
authors.initials: RA
authors.lastname: Pfuetzner
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Larson Freeman
authors.forename: Samuel I
authors.initials: SI
authors.lastname: Miller
authors.forename: Patricia
authors.initials: P
authors.lastname: Hernandez
authors.forename: Ron D
authors.initials: RD
authors.lastname: Appel
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D010455:Peptides; D011506:Proteins; D015786:Cytochromes b5; D019392:Cytochrome P-450 CYP2E1